Intervention of AKR1C3 on Malignant Biological Behavior of Breast Cancer Cells through PD1/PD-L1 Signaling Pathway
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摘要:
目的 探索酮还原酶家族1成员C3 （aldo-keto reductase family 1 member C3，AKR1C3）对乳腺癌恶性细胞生物学行为的干预作用及对程序性细胞死亡蛋白/程序性死亡-配体1 （ programmed cell death protein1/programmed death-ligand1，PD-1/PD-L ） 通路的影响。 方法 把MCF-7人乳腺癌细胞中NC组和AKR1C3组分别转染空质粒和AKR1C3质粒。采用MTT法检测转染后24 h、48 h、72 h细胞活力。采用流式细胞技术测定各组细胞的存活率以及早期、晚期凋亡比例；通过Transwell实验对各组细胞的迁移和侵袭能力进行检测；通过Western blot检测各组细胞PD-1、PD-L1、蛋白激酶B（protein kinase b，AKT）蛋白表达水平。使用C57BL/6小鼠构建荷瘤模型，将采用人乳腺癌MCF-7细胞转染NC质粒和AKR1C3质粒进行细胞荷瘤，每3天测量瘤体积，持续21 d。 结果 相较于NC组，AKR1C3组细胞活力降低（P < 0.05），并且具有时间依赖效应（P < 0.05），迁移和侵袭能力降低（P < 0.05），早期凋亡和晚期凋亡比例升高（P < 0.05），PD-1、PD-L1、AKT蛋白表达水平降低（P < 0.05）。小鼠实验结果表明，AKR1C3组小鼠肿瘤体积降低，肿瘤质量下降（P < 0.05）。 结论 AKR1C3可以抑制人乳腺癌细胞恶性生物学行为，抑制PD-1/PD-L1信号通路蛋白表达。 Abstract:Objective To investigate the effect of aldo-keto reductase family 1 member c3（AKR1C3） on malignant biological behavior of breast cancer cells and its influence on the programmed cell death protein 1/programmed death-ligand 1 （PD-1/PD-L1） pathway. Methods MCF-7 human breast cancer cells with NC and AKR1C3 groups transfected with NC plasmid and AKR1C3 plasmid respectively. Cell viability at 24 h/48 h/72 h post-transfection was assessed by MTT assay; flow cytometry measured cell survival rate and early/late apoptosis ratios; Transwell evaluated migration and invasion capabilities; western blot detected PD-1, PD-L1, protein kinase B （AKT） protein expression. For in vivo experiments, the C57BL/6 mice were used to establish tumor-bearing models. Human breast cancer MCF-7 cells transfected with NC plasmid and AKR1C3 plasmid were used for cell tumor-bearing. The tumor volume was measured every 3 days for 21 days. Results Compared to NC groups, the AKR1C3 group showed significantly reduced cell viability （time-dependent） （P < 0.05）, suppressed migration/invasion （P < 0.05）, increased early/late apoptosis ratios （P < 0.05）, and downregulated PD-1/PD-L1/AKT protein expression （P < 0.05）. In vivo, AKR1C3 group exhibited reduced tumor volume and weight （P < 0.05）. Conclusion AKR1C3 inhibits malignant biological behaviors in breast cancer cells and suppresses PD-1/PD-L1 signaling pathway protein expression. -
Key words:
- AKR1C3 /
- Human breast cancer /
- Apoptosis /
- Migration /
- PD-1 / PD-L1 pathway /
- Invasion /
- AKT /
- PPR5
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图 1 过表达AKR1C3转染效率验证（$\bar x \pm s $，n = 3）
与NC组相比，*P < 0.05。
Figure 1. Validation of AKR1C3 overexpression transfection efficiency in MCF-7 cells （$\bar x \pm s $，n = 3）
图 2 过表达AKR1C3对MCF-7细胞凋亡的影响（$\bar x \pm s $，n = 3）
Figure 2. Effect of AKR1C3 Overexpression on Apoptosis in MCF-7 Cells（$\bar x \pm s $，n = 3）
图 3 过表达AKR1C3对MCF-7细胞迁移和侵袭的影响（$\bar x \pm s $，n = 3）
Figure 3. Effect of AKR1C3 Overexpression on Migration and Invasion in MCF-7 Cells （$\bar x \pm s $，n = 3）
图 4 过表达AKR1C3对MCF-7细胞PD-1/PD-L1信号通路的影响（$\bar x \pm s $，n = 3）
与NC组相比，*P < 0.05。
Figure 4. Effect of AKR1C3 Overexpression on the PD-1/PD-L1 Signaling Pathway in MCF-7 Cells （$\bar x \pm s $，n = 3）
图 5 过表达AKR1C3对肿瘤生长的影响（$\bar x \pm s $，n = 3）
A：肿瘤代表性图片；B：肿瘤体积统计图；C：肿瘤质量统计图；D：对PD-1/PD-L1蛋白的影响；与NC组相比，*P < 0.05
Figure 5. Effect of AKR1C3 Overexpression on Tumor Growth （$\bar x \pm s $，n = 3）
表 1 AKR1C3引物序列信息
Table 1. AKR1C3 primer sequence information
基因 引物序列 引物长度（bp） AKR1C3 F 5′-CAA GCG TCC TAA TTG TGG TCA-3′
R 5′-CCC TGC TAG ATG TCC AAC TGA TT-3′22 GAPDH F 5′-AGA AGG CTG GGG CTC ATT TG-3′
R 5′ AGG GGC CAT CCA CAG TCT TC-3′20 
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表 2 过表达AKR1C3对人乳腺癌MCF-7细胞活力的影响（$\bar x \pm s $，n = 3）
Table 2. Effect of AKR1C3 overexpression on cell viability in MCF-7 cells （$\bar x \pm s $，n = 3）
组别 24 h 48 h 72 h 组内时间比较 对照组 98.65 ± 0.71 97.81 ± 0.88 96.46 ± 0.61 F=1.32，P=0.312 NC组 97.92 ± 0.91 95.61 ± 1.71 95.98 ± 1.02 F=2.15，P=0.152 AKR1C3组 77.12 ± 1.41* 70.61 ± 1.73*# 61.21 ± 1.52*# F=86.34，P < 0.001 P <0.001 <0.001 <0.001 与NC组比较，*P < 0.05；与24 h相比，#P < 0.05。
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表 3 过表达AKR1C3对人乳腺癌MCF-7细胞凋亡的影响（$\bar x \pm s $，n = 3）
Table 3. Effect of AKR1C3 Overexpression on Apoptosis in Human Breast Cancer MCF-7 Cells （$\bar x \pm s $，n = 3）
组别 细胞存活（%） 早期凋亡（%） 晚期凋亡（%） 对照组 94.51 ± 0.91 3.21 ± 0.18 1.06 ± 0.05 NC组NC 93.21 ± 0.98 3.16 ± 0.47 1.28 ± 0.52 AKR1C3组 61.71 ± 5.73* 13.21 ± 0.41* 25.21 ± 1.02* F 89.675 715.490 1320.242 P <0.001 <0.001 <0.001 与NC组比较，*P < 0.05。
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表 4 过表达AKR1C3对人乳腺癌MCF-7细胞迁移和侵袭的影响（$\bar x \pm s $，n = 3）
Table 4. Effect of AKR1C3 Overexpression on Migration and Invasion in Human Breast Cancer MCF-7 Cells （$\bar x \pm s $，n = 3）
组别 细胞迁移数（个） 细胞侵袭数（个） 对照组 297.51 ± 15.41 310.21 ± 20.11 NC组NC 293.91 ± 26.98 313.11 ± 23.47 AKR1C3组 161.11 ± 15.23* 131.27 ± 26.41* F 45.418 59.078 P <0.001 <0.001 与NC组比较，*P < 0.05。
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表 5 过表达AKR1C3对人乳腺癌MCF-7细胞PD-1/PD-L1信号通路的影响（$\bar x \pm s $，n = 3）
Table 5. Effect of AKR1C3 Overexpression on the PD-1/PD-L1 Signaling Pathway in Human Breast Cancer MCF-7 Cells （$\bar x \pm s $，n = 3）
组别 PD-1 PD-L1 AKT 对照组 1.21 ± 0.15 1.50 ± 0.43 1.50 ± 0.11 NC组 1.22 ± 0.34 1.56 ± 0.32 1.59 ± 0.30 AKR1C3组 0.81 ± 0.12* 0.65 ± 0.09* 0.79 ± 0.21* F 8.828 7.892 11.821 P 0.015 0.021 0.008 与NC组比较，*P < 0.05。
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表 6 过表达AKR1C3对肿瘤生长的影响（$\bar x \pm s $，n = 3）
Table 6. Effect of AKR1C3 Overexpression on Tumor Growth （$\bar x \pm s $，n = 3）
组别 肿瘤体积（cm3） 肿瘤质量（g） PD-1 PD-L1 AKT NC组 1.22 ± 0.34 2.36 ± 0.32 2.21 ± 0.22 1.56 ± 0.33 2.51 ± 0.34 AKR1C3组 1.01 ± 0.12* 1.15 ± 0.09* 1.21 ± 0.18* 0.78 ± 0.25* 1.11 ± 0.34* t 6.781 7.892 15.88 16.78 19.21 P 0.0078 0.0088 <0.001 <0.001 <0.001 与NC组比较，*P < 0.05。
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